Isolation and Purification of Murine Megakaryocyte-Erythroid Progenitors

Female C57BL/6 or GFP mice (8–10 weeks of age) were sacrificed, and femurs and tibiae were flushed with sterile PBS to obtain bone marrow. Subsequently, bone marrow cells were filtered through a 70 µm pore size cell strainer (BD Biosciences, Bedford, MA). Red blood cells (RBC) were lysed using a sterile lysis buffer (8.26 g/L ammonium chloride, 1 g/L sodium bicarbonate and 0.01 M EDTA). Lineage-positive (Lin + ) cells were isolated by magnetic-activated cell sorting beads (MACS) using the MagCellect mouse hematopoietic cell lineage depletion kit in accordance with the manufacturer’s protocol (R&D Systems, Minneapolis, MN)s. The Lin+ cells were stored and used separately. MEPs were isolated from the remaining Lin- cells using erythroid membrane-associated protein (ERMAP)-biotin antibody (Bioss antibodies, bs-12333R-Biotin). ERMAP-positive cells were collected and re-suspended in sterile PBS. In some experiments, Lin- cells depleted from MEPs were used. The depletion was carried out by magnetic beads conjugated with ERMAP biotinylated antibody. The cell populations were used for different purposes as described below.